Tankyrases catalyze post-translational modification of target proteins, which controls their stability (15,32). TNKS1 PARsylates tankyrase target proteins, including TRF1, centrosomal P4.1-associated protein (CPAP), AXIN, PTEN and AMOTs (15,16,21,32,33). The PARsylated protein is then recognized by the E3 ligase, and targeted for ubiquitination and proteasomal degradation (15,16,26).

Tankyrase activity is controlled by a variety of factors, including polo-like kinase-1 (PLK1) and mitogen-activated protein kinase (MAPK). TNKS1 is phosphorylated by PLK1, glycogen synthase kinase 3 (GSK3), and MAPK, although the precise functions of this modification are not clear (34–36). PLK1-mediated phosphorylation results in increased TNKS1 stability and telomeric PARP activity (34). GSK3-mediated phosphorylation of TNKS1 does not alter TNKS1 auto-PARsylation in vitro, and MAPK-mediated phosphorylation of tankyrase enhances the PARsylation activity of TNKS1 in vitro (35,36). GDP-mannose 4, 6-dehydratase binds to TNKS1, inhibits tankyrase PARP activity in vitro and influences TNKS1 stability in vivo (37). Kang et al (26) demonstrated that TNKS1 is involved with the antioxidant enzyme PrxII. PrxII is essential for full TNKS1 activity, in order to maintain oncogenic β-catenin signaling in colorectal cancer (CRC). This study demonstrated the molecular mechanisms regulating tankyrase activity in CRC for the first time.

TNKS1 has been identified as an interaction partner of TRF1 (18,38). TRF1 blocks the access of telomerase to telomeres (18,32). TNKS1-mediated PARsylation of TRF1 releases TRF1 from telomeres, and the released TRF1 is degraded by the ubiquitin-proteasome pathway (18,32). Telomere maintenance by telomerase allows continued proliferation of cancer cells and is considered as a promising target for anticancer strategies. TNKS1 controls telomerase inhibition in human cancer cells and is a potential telomere-directed anticancer target (39,40). Telomere-directed inhibitors result in progressive telomere shortening, with no acute cytotoxicity, and combination with tankyrase inhibitors has been proposed (39,40). Dual inhibition of TNKS1 and telomerase has demonstrated a synergistic effect in lung and gastric cancer cell lines (41,42). Furthermore, the combination of tankyrase and telomerase inhibition promotes human lung adenocarcinoma cell apoptosis and inhibits proliferation (41). These observations indicate that co-inhibition of telomerase and tankyrase may be an effective strategy for the treatment of lung cancer in humans.

Tankyrases have been implicated in oncogenic pathways (Wnt, YAP and AKT) (15,16,43).

The Wnt signal transduction pathway regulates numerous biological processes in diseases such as in cancer (44). AXIN is the key effector in the Wnt pathway and has been identified as a tumor suppressor. Tankyrases target AXIN for degradation, whereas tankyrase inhibitors generally stabilize it (44,45). The Wnt pathway regulates proteolysis of the downstream effector β-catenin with the β-catenin destruction complex, which includes adenomatous polyposis coli (APC), AXIN and GSK3β (45). TNKS1-mediated AXIN PARsylation induces AXIN degradation with the ubiquitin-proteasome pathway, and the ensuing AXIN degradation triggers disruption of the β-catenin destruction complex (15). Released β-catenin translocates into the nucleus and switches on Wnt-dependent transcription (44).

The tumor suppressor APC scaffolds the β-catenin destruction complex (45). APC is mutated in >80% of CRC cases (46). Therefore, due to tankyrases regulating Wnt signaling, tankyrase inhibitors may be promising therapeutic targets for CRC. TNKS1 inhibition suppresses Wnt signaling and tumor growth in APC-mutant colorectal tumors (15,47,48), and increases chemosensitivity in colon cancer cell lines (49). Due to the Wnt pathway being involved in lung cancer (50,51), antagonizing the Wnt pathway through tankyrase inhibition may be effective against lung cancer, and there is evidence for tankyrases as antineoplastic targets in lung cancer (52,53).

Croy et al (27) indicated that the fly APC homolog APC2 may be a tankyrase substrate and that tankyrases regulate destruction complex activity, providing additional insight into tankyrase inhibition as a potential Wnt-pathway cancer therapy.

The Hippo pathway controls tissue homeostasis and organ size (54,55). YAP has been identified as an oncoprotein and the key effector in the Hippo pathway (54–58). YAP signaling has also been demonstrated to be involved in human cancer types (56–58). AMOTs are negative YAP regulators (33), and recent studies indicated that tankyrase inhibition suppresses YAP oncogenic activity by stabilizing AMOTs through inhibiting their tankyrase RNF146 axis-mediated degradation (28,59). These results indicate a potential opportunity for cancer therapy. Lin et al (43) demonstrated that YAP signaling is involved in drug resistance, including with RAF- and MAPK-targeted cancer therapy. Wang et al (59) reported that tankyrase inhibition enhances epidermal growth factor receptor (EGFR) growth inhibition in non-small cell lung cancer (NSCLC). These data indicate that tankyrase inhibition could be an effective approach to overcome drug resistance for combinatorial cancer therapy.

PTEN is an important tumor suppressor, and PTEN mutations have been associated with a number of cancer types (60,61) and Cowden syndrome (62). Li et al (16) identified PTEN as a tankyrase-binding protein. PTEN stabilization by tankyrase inhibition induces downregulation of AKT phosphorylation, suppressing cell proliferation and tumor growth. These data support the therapeutic potential of tankyrase inhibitors targeting the AKT oncogenic pathway.

TNKS1 is required to resolve sister telomeres during mitosis. Sister chromatid cohesion holds sister chromatids together from their S phase replication until their mitosis separation (22,29). Cohesion requires a multi-protein complex comprising structural maintenance of chromosomes protein (Smc)1, Smc3, sister chromatid cohesion protein (Scc)1 and Scc3 (63,64). In the absence of TNKS1, cohesion is removed from arms and centromeres, but sister telomeres remain associated, indicating persistent cohesion, and this persistent telomere cohesion by TNKS1 inhibition during mitosis induces a delay in anaphase progression (22). Tripathi and Smith (29) demonstrated that the mechanism of cell cycle-regulated K63-ubiquitination of tankyrase controls sister telomere resolution timing.

TNKS1 colocalizes with the nuclear mitotic apparatus (NuMA) protein and PARsylates NuMA in mitosis (20), and TNKS1-depleted cells exhibit defects in mitotic spindle assembly and structure (19), indicating that TNKS1 is required for normal spindle formation. TNKS1 also localizes at the centrosomes, promotes centrosome maturation, interacts with CPAP, PARsylates CPAP, and regulates CPAP protein stability and function at centrosomes across the cell cycle (21). Miki PARsylation by TNKS1 promotes centrosome maturation (65); therefore, CPAP and Miki may have a general role in centrosome function. Abnormal centrosomes are involved in cancer and contribute to chromosome missegregation and aneuploidy, thereby promoting malignant progression (66–69). Korzeniewski et al (70) indicated centrosomes as a potential target for cancer therapy.

The DNA-dependent protein kinase (DNA-PK) is a critical component of non-homologous end joining-mediated DNA repair mechanisms (71). DNA-PKcs, a catalytic subunit of DNA-PK, exists in a PARsylated state in vitro and in vivo (72,73). TNKS1 regulates DNA repair via PARsylation-mediated stabilization of DNA-PK and suppression of telomere-associated sister chromatid exchange (74). Tankyrases bind to mediator of DNA damage checkpoint protein 1 and promote homologous recombination and checkpoint activation in response to double-strand breaks (DSBs) (75). Therefore, tankyrases have a direct role in DSB repair. DNA-PK is involved in tumor-associated processes, including genomic stability, hypoxia, metabolism, inflammatory response and transcription (71), which indicates that DNA-PK may be a potential target for cancer therapy.

Tankyrase inhibition blocks proliferation and promotes cell apoptosis in neuroblastoma (NB) cell lines; therefore, tankyrases are a potential target for NB (17).

PTEN has been characterized as a tumour suppressor, and PTEN mutations have been reported in cancers (60,61) and Cowden syndrome (62). Li et al (16) identified PTEN as a tankyrase-binding protein containing a RXXXDG tankyrase-binding motif (RYQEDG). Tankyrases interact with and PARsylate PTEN. PARsylated PTEN promotes PTEN degradation through E3 ligase RNF146 (16).

PTEN stabilization by tankyrase inhibition induces downregulation of AKT phosphorylation, suppressing cell proliferation and tumor growth (16). These data indicate a therapeutic potential for tankyrase inhibitors in cancer, targeting the AKT oncogenic pathway.

Kang et al (26) demonstrated that TNKS1 is involved with PrxII, and PrxII is essential for full TNKS1 activity to maintain oncogenic β-catenin signaling in CRC. In addition, it was indicated that the TNKS1 zinc-binding motif is essential for PARP activity and is protected from oxidative inactivation by PrxII. Furthermore, H₂O₂-dependent inactivation of TNKS1 PARP activity in the absence of PrxII enhances AXIN-dependent β-catenin degradation in APC-mutant CRC cells (26). These results indicate that PrxII inhibition may exert therapeutic effects on APC-mutant CRC cells.

Of all colon cancer cases, >80% are initiated by truncating mutations in the tumor suppressor APC (46). The Wnt pathway regulates proteolysis of the downstream effector β-catenin via the β-catenin destruction complex, including APC, AXIN and GSK3β (45). Croy et al (27) identified APC2, a fly APC homolog, as a tankyrase binding partner and substrate. This previous study indicated that tankyrases regulate the activity of the β-catenin destruction complex through AXIN and APC2 ribosylation, supporting the therapeutic value of tankyrase inhibition as a Wnt-pathway cancer therapy.

AMOTs are negative YAP regulators (33), and tankyrase inhibition suppresses YAP oncogenic activity by stabilizing AMOT family proteins (28,59). Tankyrases bind to AMOTs, and tankyrase-mediated PARsylation induces their degradation through E3 ligase RNF146 (28,59). These observations highlight the therapeutic potential of tankyrase inhibitors in cancer, targeting the YAP oncogenic pathway. YAP signaling has been associated with drug resistance in cancer types, including lung cancer, and hence YAP signaling inhibition is important to overcome drug resistance (84). Tankyrase inhibition enhances EGFR inhibitor growth inhibition in lung cancer cells via AMOT stabilization and YAP signaling inhibition (59). Thus, tankyrase inhibition may be an effective approach to overcoming drug resistance for combinatorial cancer therapy.

During the S phase, DNA must not only be replicated, but also newly synthesized DNA molecules must also be connected with each other (85). This sister chromatid cohesion is essential for chromosome segregation during mitosis. Canudas and Smith (86) demonstrated that premature resolution of telomere cohesion between sister telomeres induced sister telomere loss. Resolution at telomeres requires TNKS1, but TNKS1 mechanisms in timely resolution of sister telomere cohesion are poorly understood. Tripathi and Smith (29) identified ABRO1, the scaffold subunit of the BRCC36 deubiquitinating enzyme (BRISC DUB) and demonstrated that sister telomere resolution timing was ensured through cell cycle-regulated ubiquitination of TNKS1 by RNF8 ligase and the BRISC DUB. Perturbation of this regulation results in persistent unresolved cohesion in mitosis or premature loss of cohesion in the S phase, indicating that a cell cycle-regulated post-translational modification controls sister telomere cohesion timing to ensure genome integrity.

The adapter protein CD2AP is essential for kidney ultrafiltration and is expressed primarily by podocytes in the kidney. Podocyte damage results in numerous glomerular diseases, including nephritic syndrome and nephrotic syndrome (87). Kuusela et al (30) demonstrated that tankyrases interact with CD2AP, and CD2AP is a negative tankyrase regulator. Tankyrase inhibition in the absence of CD2AP increases kidney damage, which indicates that tankyrases are essential for maintaining normal kidney function (87).

Li et al (31) investigated the tankyrase protein interaction network through proteomic analysis and identified >100 high-confidence interacting proteins for tankyrases. In particular, they demonstrated that TNKS1 and TANK2 bind to the peroxisome protein PEX14 and localize on peroxisomes. Overexpression of TNKS1 or TANK2 decreases peroxisome number or size, indicating that TNKS1 and TANK2 promote pexophagy. This study also demonstrated that tankyrases associate with the autophagy-associated protein ATG9A. Additionally, they indicated that tankyrase may associate PEX14 with ATG9A to promote pexophagy. Further experimentation is required to confirm the detailed mechanism. This study provides insights into further cellular localizations and functions.